Skip to main content
Premium Trial:

Request an Annual Quote

New Approach Detects DNAse I Hypersensitive Sites Genome-Wide in Single Cells

NEW YORK (GenomeWeb) – A US National Institutes of Health-led team of researchers has devised a new approach to detect DNAse I hypersensitive sites genome-wide in single cells.

Such DHS sites mark key genomic regulatory regions and the chromatin state of cells, but conventional DNase sequencing requires millions of cells to generate genome-wide DHS profiles. Keji Zhao, a researcher at the National Heart, Lung, and Blood Institute, and his colleagues have developed a new circular carrier DNA-mediated sequencing method, called single-cell DNase sequencing or scDNase-seq, to detect DHSs in single cells. They applied the approach, reported in Nature today, to cultured cells to examine DHS patterns, as well as to cells from formalin-fixed, paraffin-embedded tumors to suss out tumor-specific DHSs.

"ScDNase-seq can reliably detect DHSs in single cells, greatly extending the range of applications of DHS analysis both for basic and for translational research, and may provide critical information for personalized medicine," the authors wrote in their paper.

Their approach consists of FACS-sorting single cells for digestion with DNase I, followed by end-repair and adaptor ligation, PCR amplification in the presence of circular carrier DNA, and sequencing.

Zhao and his colleagues applied scDNase-seq to NIH3T3 cells to create DHS profiles for sets of 10,000, 1,000, 100, and single cells. Per single cell, the researchers generated an average of 317,000 unique scDNase-seq reads and discovered 38,000 DHSs.

DHS patterns found in single cells were largely similar, the researchers found, and pooled DHSs from five NIH3T3 cells correlated with those generated from 1,000 cells. Still, while 90 percent of the DHSs uncovered in single cells could be detected in a population of cells, only between 35 percent and 59 percent of the DHSs found in a population of cells could be seen in each single cell.

In addition, by examining DHSs that are specific to NIH3T3 cells and those that are specific to embryonic stem cells, the researchers found that cell-specific DHSs largely correlated with cell-specific gene and biological functions.

As chromatin defects have been implicated in a number of cancers, the researchers applied their scDNase-seq approach to pools of cells isolated from an FFPE-preserved follicular thyroid carcinoma sample, identifying 1,342 tumor-specific and 2,812 normal-specific DHSs. The tumor-specific DHSs were enriched for sites involved in processes like the regulation of GTPase activity and hypoxia response, and in the E-cadherin signaling, RhoA signaling, p53 pathway, RAC1 signaling, and MYC transformation pathways.

The researchers noted that genes characteristic of a PAX8-PPARG fusion in FTC were enriched in tumor-specific DHSs — even if the rearrangement was not detected by FISH. This, they added, indicates that pathways associated with transcriptional regulation by PAX8-PPARG — and perhaps not the PAX8-PPARG rearrangement itself — are important for FTC development.

Zhao and his colleagues further applied their approach to two additional FTC samples and a papillary thyroid carcinoma. They noted that the three FTC samples shared few DHS sites. This and other findings, they said, suggest that most DHSs are patient-specific and that tumors may develop and progress through different mechanisms in different patients.

For instance, in one FTC patient, they uncovered 31 SNVs in DHS regions, including a de novo mutation at a DHS downstream of the thioredoxin-like 1 (TXNL1) gene.

TXNL1, the researchers noted, encodes a regulatory subunit of the human 26S proteasome, and its downregulation has been linked to poor outcomes, aneuploidy in colorectal carcinoma, and cisplatin-induced apoptosis.

This particular variant seems to affect the p53-binding motif and is correlated with decreased TXNL1 expression in tumor cells, the researchers added.

Overall, the approach could be a cost-effective alternative to genome sequencing to uncover functionally important mutations in regulatory regions.

"Being able to evaluate the chromatin states associated with specific diseases or developmental programs might provide valuable information for developing new diagnostic and therapeutic strategies for these malignancies," Zhao and his colleagues added.